A UPLC-MS/MS method for simultaneous determination of arachidonic acid, stearic acid, and related endocannabinoids in human plasma

Endocannabinoids (eCBs) exert considerable influence over energy metabolism, lipid metabolism, and glucose metabolism within the human body. Among the most biologically active cannabinoids identified thus far are 2-arachidonoylglycerol (2-AG), arachidonoyl ethanolamide (AEA), 1-stearoylglycerol (1-SRG), and stearoyl ethanolamide (SEA), which are derived from arachidonic acid (AA) and stearic acid (SA). However, despite the unique in bioactivities exhibited by eCBs, their determination in plasma has been hindered by the lack of sensitive analytical methods. The aim of this study was to develop and validate a highly sensitive and rapid method using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for accurate measurement of AEA, SEA, 2-AG, 1-SRG, AA, and SA levels in human plasma samples. Sample preparation involved a protein precipitation method and a methyl tert-butyl ether liquid-liquid extraction method. Chromatographic separation was accomplished by utilizing an ACQUITY UPLC BEH C8 column with a mobile phase of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid, flowing at a rate of 0.35 mL/min. AA-d8, 2-AG-d5, and AEA-d8 were selected as deuterated internal standards. The analytes were determined with MRM in both positive and negative ion mode. The lower limit of quantification ranged from 0.1 to 400 ng/mL, and the correlation coefficient (R2) was >0.99. Inter-day and intra-day precision exhibited values of 0.55–13.29% and 0.62%–13.90%, respectively. Recovery and matrix effect were within the range of 77.7%–109.7%, and 90.0%–113.5%, respectively. Stability tests confirmed the acceptability of all analytes. To demonstrate the effectiveness of the approach, it was implemented to assess and compare plasma samples from healthy volunteers (n = 49) and individuals with non-alcoholic fatty liver disease (NAFLD) (n = 62). The study revealed significant differences in AEA, SEA, AA, and SA levels between the two groups.